The U.S. government has rights in this invention by virtue of Grant No. NIH-5-R01-AI17879-02 of the National Institute of Health.
This invention relates to methods of efficiently expressing DNA introduced into eucaryotic cells. More specifically, the invention relates to a method of exploiting the genetic mechanism of certain types of eucaryotic cells to produce relatively large quantities of a protein of interest or its precursor.
The protein production of most animal cells is limited to synthesis of enzymes used by the cell for metabolism and to structural proteins, surface proteins, and numerous proteinaceous materials having specialized functions such as interferons, lymphokines, and hormones. Typically, such cells produce relatively modest amounts of these proteins. Other types of cells in addition are capable of producing and secreting large amounts of proteinaceous material for systemic use in the animal body. Examples of the latter type of cells include cells of the circulatory system which produce globulins and fibrinogen, liver cells which produce serum albumin, and the beta cells of Islets of Langerhans which produce insulin. If the genetic mechanisms responsible for the high level production could be used to produce lymphokines, interferons, antibodies, or other proteinaceous materials of interest, large supplies of valuable proteins could be made available.
Hybridoma technology in effect harnesses the proteinproducing capabilities of a B cell to produce monoclonal antibodies and accordingly achieves this goal to some extent. To produce hybridomas, a lymphoid cell that has been stimulated to produce antibody by in vivo or in vitro immunization is fused with an immortal cell line, for example, a myeloma cell line. The fusion products are grown in a selective medium in which the parental myeloma cannot survive and then screened for a clone which secretes the monoclonal antibody of interest. Using this technique, one may obtain monoclonal antibodies of high affinity for specific antigens. However, no reproducible method has been developed which permits application of the hybridoma technology to the production of non-immunoglobulin proteins. Furthermore, certain types of potentially useful hybridoma cells, e.g., human x human hybridomas, are notriously difficult to culture.
De Villiers et. al., in Nucleic Acid Research, Volume 9 No. 23, pg. 6251, 1981, disclose work involving the linking of a rabbit hemoglobin gene with a 244 base pair DNA fragment derived from the beginning of the polyoma virus late region. After transfection of such recombinant DNA into mouse 3T6 and human Hela cells, the polyoma sequences were found to strongly enhance the level of correct beta globin gene transcripts over a distance of at least 1400 base pairs. The authors hypothesized that this 244 base pair DNA fragment of viral origin, termed an "enhancer", might be useful as a component of mammalian expression vectors. Conrad et. al., in Molecular and Cellular Biology, pages 949-965, August, 1982, disclose that a recombinant library of human DNA sequences was screened with a segment of simian virus 40 DNA that spans the viral origin of replication. One SV40 hybridized fragment contained a sequence which increased the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. The authors reported that this effect was orientation independent when the sequence was present at the 3' end of the chicken thymidine kinase gene and proposed that this segment of DNA contains a sequence analogous to the 72 bp repeats of SV40. However, no direct evidence was presented indicating this element is a transcription enhancer. For example, the activity seen by these authors could result from increased integration of the introduced thymidine kinase gene into the host DNA. Weiher et. al., in Science, Volume 219, page 626, Feb. 11, 1983, disclose that viral enhancers can stimulate transcription from heterologous promoters and that such enhancers have been found in papovaviruses and retroviruses, e.g., SV-40, polyoma, Moloney sarcoma and Bovine papilloma.